1. Field of the Invention
The present invention relates generally to the field of cellular regulation. More particularly, it concerns the control of cell growth as affected by the retinoblastoma protein, the implications of this control in cancers including retinoblastoma, and the interactions of oncogenes with cell cycle control proteins and other retinoblastoma associated cellular proteins.
2. Description of the Related Art
Retinoblastoma is a malignant cancer of the developing retina that manifests itself as an intraocular tumor in early childhood (Shields, J. A., 1983). Retinoblastoma occurs in two distinct forms (Vogel, F., 1979). In close to 40% of retinoblastoma cases, tumors appear bilaterally, may be multifocal in each eye, and appear at a very young age. Bilateral retinoblastoma is sometimes diagnosed in newborn infants. In the remaining retinoblastoma cases, tumors are unilateral, are unifocal, and generally present at about 2 years of age.
The pattern of inheritance of retinoblastoma suggests that the wild-type alleles encode a repressor of tumor formation (Migdal, C., 1976). Evidence for the existence of tumor-suppressing genetic factors is also provided by cell fusion studies. Upon fusion of tumor cells with normal fibroblasts, lymphocytes, or keratinocytes (Harris, H., 1986a; Klein, G., et al., 1971), the malignant properties of the original tumor cells are suppressed.
A candidate for the retinoblastoma suppressor gene (Rb) has been cloned, and extensive structural and functional analysis of the gene and its gene product has begun (Goodrich and Lee, 1993). The Rb gene product has properties that suggest a role in regulation of the cell division cycle. For example, Rb protein forms complexes with many cellular and viral proteins, including transcription factors, that influence the cell cycle. The purified Rb protein can also arrest G1 phase progression of the cell cycle when introduced by microinjection. Also, the protein is phosphorylated in a cell cycle dependent manner, suggesting that Rb may be cyclically regulated.
Deletions within the Rb gene have been defined in an extensive number of retinoblastomas (Lee, W.-H., et al., 1987a, 1987b). In addition, about 12-30% of retinoblastomas have detectable genomic rearrangement of Rb, and it is believed that other undetectable small and deletions are present but go unnoticed. In one particular study, a large number of retinoblastoma cell lines and primary tumors were screened and none were detected with normal expression of the Rb protein (Horowitz, J. M., et al., 1990). It is believed that no retinoblastoma primary tumor or cultured cell has been reported to have normal expression of the wild-type Rb gene product. In addition, mutations in the Rb gene have been implicated in other types of cancer such as osteosarcomas, and soft tissue sarcomas (Weichselbaum, R. R., et al., 1988).
An interesting biochemical property of the Rb gene product is its ability to form specific complexes with the transforming proteins of several DNA tumor viruses including SV40 large T, adenovirus E1A, and human papillomavirus E7 (DeCaprio, J. A., et al., 1988; Dyson, N., et al., 1989a; Whyte, P., et al., 1988). The regions of the transforming proteins required for complex formation with Rb are similar to those required for transformation of cells. The amino acids of Rb protein required for binding to SV40 T antigen have also been determined (Huang, S., et al., 1990), and the regions of these amino acids correspond to those frequently mutated in tumor cells. For example, in all cases analyzed to date, mutated Rb proteins from human tumor cells have been unable to form complexes with T antigen. This correlation between the T antigen binding domains and the naturally occurring mutations in cancer cells suggests that these regions constitute an important functional domain.
It is suggested that DNA tumor viruses induce transformation by binding a more active, unphosphorylated form of the Rb protein, thereby inhibiting its normal, suppression function (Ewen, M. E. et al., 1989). The mechanism of transformation by DNA tumor viruses, however, may be more complex than simple inactivation of Rb. Several reports have indicated that binding Rb protein is not sufficient to induce the full transformation potential of some DNA tumor viruses (Manfredi and Prives, 1990; Weber, J. M. et al., 1991). However, binding of DNA tumor virus transforming proteins has pinpointed a region of Rb that is required for protein association and may be important for its normal function.
What is needed, then is a method of regulating cell growth and blocking tumorigenesis through the control of tumor suppressor proteins in their interaction with oncogene products. This control is possible through the interaction of these tumor suppressors and the cellular proteins that bind at functional sites and modulate their biological activity.